1. Pathogenic Interplay between LRRK2 and Tau in PD. To examine a potential pathogenic interplay between LRRK2 and tau in neurodegeneration, we crossbred CaMKII-tTA/tetO-G2019S LRRK2 double transgenic mice with a line of tau transgenic mice Prnp-tau B6D2-Tg(Prnp-MAPT)43Vle/J to generate CaMKII-tTA/tetO-G2019S/Prnp-tau triple transgenic mice that co-expressed human LRRK2 and tau in neurons of forebrain regions such as cerebral cortex, striatum and hippocampus. We found that co-expression of human G2019S LRRK2 and wild-type tau further decreased the level of free tubulin in the brain homogenate. Meanwhile, the formation of tau-positive aggregates was increased in the cortex of CaMKII-tTA/tetO-G2019S/Prnp-tau triple transgenic mice as revealed by immunostaining with antibodies against tau. These preliminary data indicate that LRRK2 may regulate the function and aggregation of tau and a synergistic interaction between LRRK2 and tau may further impair the microtubule dynamics in neurons. To further investigate whether LRRK2 and tau act synergistically in the midbrain dopaminergic (DA) neurons, we have generated a cohort of PITX3-tTA/tetO-LRRK2 WT/tetO-Tau WT transgenic mice in which wild-type human LRRK2 and tau are selectively expressed by the midbrain DA neurons. In the coming fiscal year, we will carry out behavioral, pathological, neurochemical studies of these mice. Meanwhile, we will also examine the subcellular phenotype in the affected DA neurons, particularly on the axonal transport and dopamine release. 2. Pathogenic Interplay between alpha-synuclein and LRRK2 in PD. We have previously shown that over-expression of LRRK2 exacerbates PD-related A53T alpha-synuclein-mediated neurodegeneration in the forebrain regions;while inhibition of LRRK2 expression ameliorates the alpha-synuclein-induced neuronal loss. However, it remains unclear whether LRRK2 and alpha-synuclein act the same way in the midbrain DA neurons. To address this question, we generate PITX3-tTA/tetO-LRRK2/tetO-alpha-synuclein triple transgenic mice to allow a selective expression of human wild-type as well as PD-related mutant LRRK2 (G2019) and alpha-synuclein (A53T) in the midbrain DA neurons. In addition, we also bred PITX3/A53T mice in the LRRK2 knockout background. We have systematically examined the numbers of midbrain DA neurons in the alpha-synuclein and LRRK2 mutant mice at one and eighteen months of age. Our preliminary data indicate that over-expression of both wild-type and G2019S LRRK2 accelerated A53T alpha-synuclein-mediated DA neuron loss in 1-month old mice. By contrast, loss of LRRK2 ameliorated A53T alpha-synuclein-induced DA neurodegeneration. In the coming fiscal year, we will generate a new cohort of wild-type LRRK2 and alpha-synuclein transgenic mice in order to investigate if the up-regulation of both wild-type alpha-synuclein and LRRK2 is sufficient to cause DA neuronal loss, and the underlying pathogenic mechanisms. This study may provide a useful animal model for studying the cause and treatment of sporadic PD. 3. Pathogenic Interplay between alpha-synuclein and tau in PD. It has been shown previously that over-expression of alpha-synuclein and tau greatly accelerate their aggregation in vitro. We decided to evaluate the pathogenic consequence of alpha-synuclein and tau interaction in the midbrain DA neurons. Together with Dr. Xian Lin's lab, we will generate PITX3/alpha-synuclein/tau triple transgenic mice to study the potential pathogenic interplay between alpha-synuclein and LRRK2 using the same strategy as we outlined the above. 4. Pathogenic Interplay between Wild-type alpha-synuclein, LRRK2, and tau in PD. Finally, we plan to develop PITX3/alpha-synuclein/LRRK2/tau compound transgenic mice to investigate the contribution of these three genetic risk factors in the degeneration of midbrain DA neurons. This study may provide a useful animal model for studying the cause and treatment of sporadic PD.